Legionella pneumophila (Lp) is the primary agent of Legionnaires'disease, a common and potentially fatal form of pneumonia. The bacterium is ubiquitous in natural and man-made water systems and infects the lungs after the inhalation of contaminated aerosols. In water and the lungs, Lp grows as an intracellular parasite, infecting either aquatic protozoa, macrophages, or epithelia. Previously, we determined that Lp possesses a type II secretion (T2S) system and hypothesized that proteins secreted by T2S are mediators of environmental persistence, intracellular infection, and virulence. During the last grant period, we documented that T2S is required for intracellular infection of amoebae, alveolar macrophages, and lung epithelia as well as bacterial persistence in the lungs of experimentally-infected mice. Proteomic analysis of Lp supernatants and in silico analysis of the Lp genome were then combined to reveal the extensive nature of the T2S output. In addition to predicted activities, which we confirmed, "novel" exoproteins were identified that had no similarity to known proteins. Using mutants lacking one or more T2S-dependent exoproteins (effectors), we determined that a metalloprotease (ProA) and ribonuclease (SrnA) promote amoeba infection, phospholipases C (PlcA/B) and a chitinase (ChiA) facilitates infection of mammalian cells, and a novel protein (Lpg0264) promotes both amoebal and macrophage infection. In vivo competition assays then identified four T2S effectors that appear to promote Lp survival in lungs;i.e., ChiA, PlcA/B, Lpg0264, and an astacin-like protease (LegP). Additionally, we discovered that Lp exhibits a swarming phenotype on agar surfaces that is dependent upon T2S. In sum, Lp T2S is uniquely critical for intracellular infection, swarming, virulence, as well as low-temperature survival in water, elaborating more effectors and encoding a wider variety of activities than is appreciated for any other T2S system. In the current proposal, we will use genetics and various infection models and cell biological tools to i) determine the importance of a select number of new "novel" effectors for intracellular infection, ii) determine the importance of T2S-dependent swarming for intracellular infection, iii) determine the intracellular location of and trafficking patterns influenced by ProA, SrnA, PlcA/B, and Lpg0264, and iv) confirm the importance of PlcA/B, Lpg0264, and LegP, the new novel effectors, and swarming in a murine model of pneumonia. The proposed studies will i) increase significantly our understanding of the pathogenesis of Lp, which is an important public health concern within the US and throughout the world, ii) expand our molecular understanding of both bacterial protein secretion and intracellular infection, iii) have implications for other important, environmental pathogens that utilize or are predicted to use T2S, and iv) offer potential new targets for disease diagnosis, treatment, or prevention. PUBLIC HEALTH RELEVANCE: We have discovered that the so-called type II secretion system of Legionella pneumophila promotes bacterial growth in mammalian host cells (e.g., macrophages and epithelial cells) and in the lungs of experimentally infected mice. We therefore hypothesize that the proteins (effectors) secreted by the bacterium through this system are virulence factors and therefore are potential targets for disease diagnosis or prevention. To test this hypothesis, we will i) characterize bacterial mutants that are lacking specific effectors using in vitro models of intracellular infection and an animal model of pneumonia in order to identify those effectors that promote infection, and ii) monitor the intracellular expression patterns of those proteins that are shown to be important for infection.